MICROALGAE – CULTIVATION · PDF fileMICROALGAE – CULTIVATION METHODS*...

POLISH JOURNAL OF NATURAL SCIENCESAbbrev.: Pol. J. Natur. Sc., Vol 27(2): 151–164, Y. 2012

MICROALGAE – CULTIVATION METHODS*

Marcin Dębowski, Marcin Zieliński, Mirosław Krzemieniewski,Magda Dudek, Anna Grala

Department of Environmental Protection EngineeringUniversity of Warmia and Mazury in Olsztyn

K e y w o r d s: microalgae, cultivation, open ponds, photobioreactors, dark systems.

A b s t r a c t

Investigations into the use of algae for energy production have been carried out for many years.A key issue is the selection of technology for culture and acquisition of algae biomass for energeticpurposes. The proliferation and culture of algae may be conducted with a variety of methods,beginning from strictly monitored methods in closed laboratory systems, to less predictable methodsin open systems. Though many systems have been developed so far, unfortunately none of them maybe found cost-effective. Photobioreactors are expensive and require high exploitation inputs (lighting,supply of carbon dioxide), they additionally pose some difficulties in exploitation e.g. due toovergrowing and restricted light penetration. In contrast, some technological systems are applied inthe technical scale that merge certain elements of open and closed systems. There is also a possibilityof algae biomass proliferation and culture in dark systems.

MIKROALGI – METODY HODOWLI

Marcin Dębowski, Marcin Zieliński, Mirosław Krzemieniewski, Magda Dudek,Anna Grala

Uniwersytet Warmińsko-Mazurski w OlsztynieKatedra Inżynierii Ochrony Środowiska

S ł o w a k l u c z o w e: mikroglony, hodowla, stawy otwarte, fotobioreaktory, systemy ciemne.

Address: Marcin Dębowski, University of Warmia and Mazury, ul. Romana Prawocheńskiego 1,10-719 Olsztyn, Poland, phone: + 48 (89) 523 41 24, e-mail: [email protected].* This research was carried out under the Key Project No. POIG.01.01.02-00-016/08 titled: Modelagroenergy complexes as an example of distributed cogeneration based on local and renewable energysources. Project financed under the OP Innovative Economy.

A b s t r a k t

Badania nad możliwością wykorzystania glonów do celów energetycznych prowadzone są odwielu lat. Jedną z najważniejszych kwestii, warunkujących opłacalność produkcji glonów, jest wybórodpowiedniego systemu hodowlanego. Stosowane są zarówno reaktory zamknięte, jak równieżtechnologie oparte na systemach otwartych. Testowane w warunkach laboratoryjnych oraz w skalitechnicznej rozwiązania technologiczne są zwykle mało opłacalne. Fotobioreaktory są drogie i stwa-rzają trudności eksploatacyjne, np. ze względu na zarastanie oraz ograniczenie dostępu światła.Systemy otwarte charakteryzują się niską efektywnością i produktywnością. W skali technicznejstosowane są również rozwiązania, które łączą pewne elementy systemów otwartych i zamkniętych.Istnieje również możliwość namnażania biomasy glonów w systemach ciemnych.

Introduction

Investigations into the use of algae for energy production have been carriedout for many years. The first attempts to culture and exploit algae for fuelproduction purposes have been undertaken during the II World War byGerman researchers. The possibility of using algae biomass in processes ofmethane fermentation was discovered in the fifties of the XXth century by,among others (Algae culture. 1953). In the sixties, algae biomass productionhas been started in a technical scale in open systems. In the eighties the USADepartment of Energy has initiated a research on the use of algae in energyproduction (Aquatic Species Program). In the successive years technologieshave been developed for biodiesel production and algae culture in photo-bioreactors, and commercial bio-refineries have been established in, amongothers, Turkey and the United States. Nowadays, a number of research andimplementation programs have been underway worldwide, including the EUMember States, that are aimed at boosting the effectiveness of algae biomassproduction and conversion into biofuels (LO et al. 2010, MUSSATTO et al. 2010,VIJAYARAGHAVAN 2009). A few thousands of patents linked with technologies ofalgae biomass production, separation and conversion into biofuels are regis-tered annually, which indicates a great global interest of scientists in thisrespect (GALLAGHER 2011, STEPHENS et al. 2010).

A key issue is the selection of technology for culture and acquisition of algaebiomass for energetic purposes. The proliferation and culture of algae may beconducted with a variety of methods, beginning from strictly monitoredmethods in closed laboratory systems, to less predictable methods in opensystems (MOLINA-GRIMA 1999).

Owing to the site the process is being run at, systems are divided into:– open systems (outdoor systems), making use mainly of open aquifers, ponds,

though under favorable environmental conditions (light exposure and hightemperature) they may also refer to photobioreactors (PBRs). Applied

Marcin Dębowski et al.152

mainly for economic concern, they provide no possibility for monitoringconditions of the culture process, and are sensitive to environmental pollu-tion, predators and competitive species. The open systems include(BOROWITZKA 1999):– traditional ponds, ground or concrete with large (up to 250 ha) area and

depth of up to 0.5 m;– circular ponds with mechanical or convective mixing;– race track-type ponds with a paddle wheel;– cascade ponds.

– closed systems, making use mainly of various types of photobioreactors orsmall tanks. Such cultures assure, most of all, the possibility of constantmonitoring over lighting and temperature, protection against predators,parasites and competitive species of algae. The closed systems include:– the sack system of “large bags”, operating in a sequential or semi-

continuous mode,– tubular photobioreactors with horizontal or vertical orientation, or in-

clined at any angle. A technological solution of this type may be arrangedin parallel or spirally as the Biocoil type reactor,

– plate photobioreactors.Owing to the duration and method of running a culture, systems may be

divided into:– batch systems, where the culture is inoculated in a single dose into the

culture medium and cultivated until the moment when the population ofcultured organisms reaches its maximum or when cell density in the cultureapproximates the maximum. Then harvest occurs, and the cultured biomassis separated from the culture medium;

– continuous systems with continuous inflow and outflow of culture mediumand continuous reception of biomass produced;

– semi-continuous systems with partial reception of biomass and continuousaddition of culture media so as to maintain algae growth rate close to themaximum.

Open systems

A very important issue is the choice of technology for culture and produc-tion of algae biomass for energy purposes. Owing to economic concerns, thesystems used currently in the industrial scale are open ponds. In most cases,their design is very simple. These are usually ground tanks with a large surfacearea and depth of up to 0.5 m, stirred mechanically by means of a paddleagitator. They are built in the form of a round pond or a racetrack. The culture

Microalgae – Cultivation Methods 153

media applied usually include chemical substances or sewages containingappropriate quantities of biogenic compounds, if necessary supplemented withmicroelements. Carbon dioxide is acquired directly from atmospheric airthrough simple diffusion. An advantage of this technological solution is simpleand inexpensive construction, its drawbacks however include high water lossesas a result of evaporation, low yield of biomass production, limited possibilitiesof culture of specific algae species susceptible to various infections, diseasesand parasites. Systems of this type prove successful in regions with highinsolation and unlimited access to water, hence at the seaside areas. Thesetechnologies are commonly applied in the Asian countries, Mexico, the USA(e.g. in Arizona) as well as in Europe, including e.g. Italy, Spain and even theNetherlands, mainly for cultures of Spirulina and Chlorella genera algae(DEMIRBAS et al. 2011).

Due to the very good results are obtained as breeding ponds in a race trackthat we decided to just describe the type of open systems. This type oftechnology not only high productivity but also is probably the most reasonablechoice in economic terms.

The system consists of shallow open ponds racetrack type with a width of2.0–3.0 m and depth 0.1–0.3 m, made of PVC, clay or paved area from 1000 to5000 m2 (MOLINA-GRIMA 2003). Pond type of race track design is based on

Fig. 1. Rays ways reactor: 1 – discharge of biomass; 2 – puddlewheel; 3 – nutrient dosing pomp;4 – medium tank; 5 – baffle


a series made in a closed loop recirculation duct. Excitation processes of mixingand flow in such a device there, using the paddle wheel. Channels of the pondcan be constructed of concrete or compressed earth and are lined with whiteplastic. The medium can be fed into the system on a continuous basis duringthe day when the culture is efficiently radiated. Nutrients are administeredbefore the paddle wheel, where the stream begins breeding. Grown biomass isreceived from the device before the paddle wheel at the end of the loop. Toprevent falling of algae stirrer is maintained constantly in motion. Rays waysreactor is shown in Figure 1.

If the wastewater system are introduced, it can remove up to 35 g BZT/m2

d (175 g BZT/m3 d pond with a depth of 0.2 m) compared to 5–10 g BZT/m2

d (5–10 g BOD/m3 d pond at a depth of 1 m) achieved in conventionalstabilization ponds (RACAULT and BOUTIN 2005). This project also requiresa much shorter hydraulic retention time (HRT) in the system, amounting to2–6 d (MARA and PEARSON 1998) compared to 10–40 d in traditional ponds(CRITES, TCHOBANOGLOUS 1998). Despite a much better cleaning efficiency, fewsuch systems are currently used for wastewater treatment in the world.

Photobioreactors

A completely different approach to the problem of proliferation and cultureof alga biomass is the application of closed systems, the so-called photobioreac-tors. A variety of these systems have been developed so far, including:horizontal tubular photobioreactors, horizontal tubular photobioreactors orsloping under any angle, biocoil type reactors, continuous or semi-continuousbig bag systems, or flat-plate photobioreactors (BOROWITZKA 1999, AMIN 2009) –Figure 2.

Photobioreactors are much more universal devices that may be appliedunder various climatic conditions. The closed character of bioreactors restrictsevaporation, eliminates the problem of parasites and predators, whilst ar-tificial lighting assures optimal conditions for photosynthesis. Such conditionsafford the possibility for running cultures of specific algae species, e.g.these with a high concentration of oil in biomass (CUARESMA et al. 2011,DEMIRBAS 2011).

The first closed photobioreactor in use was a big gags photobioreactor(BAYNES et al. 1979, WATSON 1979). It consisted of large sterile plastic bags ca.0.5 m in diameter with an adjusted aeration system. Most of those systemshave been designed to operate in the batch mode, however semi-continuoussystems do happen as well. This variant of the system was developed by COHEN

and ARAD (1989) and its modification consisted in using bags with a smaller


Fig. 2. Plate photobioreactor: 1 – medium tank; 2 – nutrient dosing pomp; 3 – air pomp; 4 – lightsource; 5 – discharge of biomass

Fig. 3. Big bags photobioreactor: 1 – medium tank; 2 – nutrient dosing pomp; 3 – gas outlet; 4 – lightsource; 5 – discharge of biomass

diameter. So far, however, a few companies have been exploiting this system(Figure 3).

The key problem linked with its exploitation is the necessity of running theprocess indoor, for there is no possibility of controlling temperature.In addition, a relatively large diameter of the bags poses problems with culturelighting, which in turn constitutes a factor diminishing system’s productivity.


A reactor of this type requires high workload and does not assure thoroughmixing, which may cause the collapse and reduction of process effectiveness.

Most of tubular reactors are made of glass or polycarbonate (PC), and theflow of medium and supply of gases proceed through pumps or, preferably, bymeans of the airlift system. They may be constructed in the horizontal(MOLINA et al. 2001), vertical or inclined (TREDICI and ZITTELLI 1998, UGWU etal. 2002), and conical orientation (WATANABE and SAIKI 1997). The aerationand mixing of culture in a tubular photobioreactor is usually conducted withthe use of air pumps or the airlift system. This reactor may be exploited alsooutdoor, as it possesses a vast illumination surface area. In contrast, one of itsmain drawbacks is a low mass transfer. This phenomenon occurs as a resultof an increasing level of oxygen along with increasing sizes of this typeof reactors. Investigations have shown that a very high level of dissolved oxygen(DO) may easily be reached in the tubular reactors (MOLINA et al. 2001).Furthermore, of key significance is the process of photoinhibition ongoing inthe tubular reactor under external (outdoor) conditions (VONSHAK and TOR-

ZILLO 2004). Once the system is scaled up by increasing tubes’ diameter, theratio of illumination surface area to system volume is decreasing. In this case,the cells at the lower part of the tube will not receive enough light for cellgrowth (due to light shading effect) unless there is a good mixing system. Then,the effectiveness of providing light to cells may be achieved through theimprovement of the mixing system (UGWU et al. 2003, UGWU et al. 2005).

An additional difficulty in a tubular photobioreactor is temperature con-trol. Though it is feasible to apply a thermostat, it is an expensive and difficultto implement solution. Worthy of notice is also the possibility of adherenceof algae cells to the walls of the reactor. In addition, a long tubular reactor ischaracterized by gradients of oxygen and CO2 transported alongside the tubes(CAMACHO RUBIO et al. 1999, UGWU et al. 2003). An increase in pH value wouldlead to the necessity of frequent re-carbonization, which in turn would increasealgae production expenditures.

A Biocoil type photobioreactor is a tubular photobioreactor composedof a transparent, plastic tube with a small diameter (2.4–5.0 cm), which isscrew-wrapped around a vertical tube with a large diameter (BOROWITZKA

1999). A few parallel systems of tubes are coupled through collectors witha pumping system that may be realized through the airlift system or a varietyof pumps. The type of the pump applied depends on algae type. The reactormay be equipped in a gas exchange system. Temperature may be controlledmanually or automatically. Reactor’s design assures uniform mixing andminimizes the adherence of algae cells to the internal walls of the tubes.Reactor’s operation may be fully automated, which enables reducing costsof the production process. The system may also be designed so as to assure theaxenic conditions (Figure 4).


Fig. 4. Biocoil photobioreactor: 1 – medium tank; 2 – nutrient dosing pomp; 3 – discharge of biomass;4 – circulation pump; 5 – light source; 6 – air pomp

Fig. 5. Column photobioreactor: 1 – medium tank; 2 – nutrient dosing pomp; 3 – air pomp; 4 – lightsource; 5 – discharge of biomass; 6 – gas outlet

Various configurations of vertical-column photobioreactors have been ex-tensively studied in view of their applicability for cultivation of algae (CHOI etal. 2003, VEGA-ESTRADA et al. 2005, GARCIA MALEA LOPEZ et al. 2006, KAEWPIN-

TONG et al. 2007). They are compact, low-cost, and easy to operate monosepti-cally (SANCHEZ MIRON et al. 2002). In addition, they are very promising for


large-scale cultivation of algae. Studies have shown that bubble-column andairlift photobioreactors (up to 0.19 m in diameter) may attain the finalbiomass concentration and specific growth rate that are comparable to valuestypically reported for narrow tubular photobioreactors (SANCHEZ MIRON et al.2002). Some bubble column photobioreactors are either equipped with twodraft tubes or constructed as split cylinders. In the first case, the mixingoccurs between the riser and the downcomer zones of the photobioreactorthrough the walls of the draft tubes (Figure 5).

Some photobioreactors may be equipped in external lighting with fluor-escence lamps or light-emitting diodes (LED), which has become the focus ofinterest in recent studies (WANG et al. 2007). Those reactors are alsoequipped in stirrers to assure the transfer of algae cells in the whole volumeof the system. Air and CO2 are supplied to the reactor from the bottom, so asto assure the maximally long time of algae cells contact with gases. This typeof photobioreactor may also be equipped in automated sensors measuring theintensity of lighting, to enable the exploitation of solar energy of both naturaland artificial origin (OGBONNA et al. 1999). In this case, the artificial source ofsolar energy is switched on only at diminished intensity of the natural source(in cloudy whether or at night). There is also a possibility of applying lightpipes for distribution of radiation in cylindrical photobioreactors (MAT-

SUNAGA t al. 1991). One of the main advantages of this type of bioreactors isthe feasibility of hot- or pressure-sterilization of their contents. Furthermore,solar energy may be supplied to the reactors in a continuous mode (both indaytime and at night) by coupled application of artificial and natural sourcesof light.

Though many systems have been developed so far, unfortunately noneof them may be found cost-effective. Photobioreactors are expensive andrequire high exploitation inputs (lighting, supply of carbon dioxide), theyadditionally pose some difficulties in exploitation e.g. due to overgrowing andrestricted light penetration. In contrast, some technological systems areapplied in the technical scale that merge certain elements of open and closedsystems. For instance, in Turkey the racetrack-type ponds were located ingreenhouses, which has a positive impact on reduced evaporation and restric-ted access of predators, enables achieving temperature stability and applyingadditional lighting if necessary. It also affords the possibility of introducingan additional source of CO2 in the form of e.g. combustion gases, to thegreenhouse’s interior. It seems that these types of solutions may provesuccessful also in Poland (UGWU et al. 2008). The table 1 shows advantagesand disadvantages of commonly used systems for the cultivation of algae.


Table 1Advantages and disadvantages of commonly used systems for the cultivation of algae

Type of system Advantages Disadvantages

round ponds,racetrack-type ponds

relatively economical, easyto use and clean after com-

Open pletion of culture, good forsystems the cultivation of algae on

a large scale

low ability to control cul-ture conditions, difficultywith cultivation algae inthe long term, low produc-tivity, large size, limitednumber of culture species,cultivation susceptible toexternal factors (pred-ators, disease, pollution)

photobioreactors column(vertical)

the high mass transfer,good mixing and lowstress, low energy con-sumption, high potentialfor scalability, easy to en-sure sterility, good for theimmobilization of algae,reduce photoinhibitionand photooxidation

a small area of exposure,their construction requiresthe use of sophisticatedmaterials, the possibilityof hydrodynamic stress,decrease in surface expo-sure with increasing diam-eter of the column

Closed photobioreactors plate the large surface area ex-systems posure, suitable for out-

door culture, good for theimmobilization of algae,good availability of light,good productivity, bi-omass, relatively cheap,easy to clean, low concen-tration of oxygen

increasing the size of thereactor requires the use ofmultiple chambers andsupporting structures,the problems of controllingthe culture temperature,the risk of fouling thewalls, the possibility of hy-drodynamic stress in somespecies of algae

horizontal tubularphotobioreactors

the large surface area ex-posure, suitable for out-door culture, good produc-tivity, biomass, relativelycheap

fluctuations in pH, dissol-ved oxygen and CO2 in thepipe length, the riskof fouling the walls, re-quires a large surface

Technological parameters of autotrophic cultivation

Irrespective of the fact whether algae biomass is produced in open systemsor in closed photobioreactors, appropriate technological parameters ought tobe assured that determine fast development and growth of the algae biomass(COHEN 1991). For optimal growth algae need appropriate lighting at the levelof 200–400 fmol photons m–2 s–1, which corresponds to ca. 1/10 of the lightingprovided directly from the Sun. Under conditions of intensive culture, thequantity of CO2 that has to be supplied to the system reaches ca. 1.83 kg per1 kg of produced biomass. In this case, wastewaters, fresh or salty water


supplemented with nitrate and phosphate fertilizers may serve as the culturemedium. Significant microelements in such a culture include nitrogen, phos-phorus, iron and in the case of selected species also silicon. Unlike that ofnitrogen and phosphorus, the availability of carbon rarely suppresses thegrowth of algae. Production performance in fresh waters is often diminished bythe availability of phosphates (CHISTI 2007). In turn, the availability of siliconis a factor reducing the growth of diatoms. A number of algae species requirean external source of vitamins, often thiamine, biotin, B12 and riboflavin,purins, pyrimidine and other growth factors. The temperature of the cultureshould oscillate in the range of 20–30oC, whereas pH value in the range of 6 to8. Water saturation with oxygen should not exceed 400% owing to thearrestment of photosynthesis and ongoing processes of photoinhibition.Worthy of notice is also that the life cycle of algae used for biofuels productionreaches 7 days (TAMBURIC et al. 2011).

Dark systems

There is also a possibility of algae biomass proliferation and culture in darksystems (BOUARABA et al. 2004) (Figure 6). These are the so-called hetero-trophic cultures, in which appropriate carbonic compounds are fed to bioreac-tors as a culture medium for the algae. Such cultures are run with acetate orglucose as a source of carbon. This system has for the first time been used inChlorella culture, which was described by KAWAGUCHI and SOONG in 1980. Asreported by LEE (1997), in the year 1996, approximately 550 t of this alga wereproduced in Japan. The Martek Inc. company (USA) runs a heterotrophicculture of Crypthecodinium cohnii to be used for the production of long-chainunsaturated fatty acids (KYLE et al. 1998). Typical conditions of the hetero-trophic culture include:– temperature: 26–28oC;– no light;– agitation with the rate of 200–480 rpm;– pH from 6.1 to 6.5;– culture medium containing ca. 20 g of glucose or acetate/L of culture;– necessary supply of nitrogen and phosphorus compounds in a quantitative

C:N:P ratio of 9:1.25:1.25.A culture in the heterotrophic system has several advantages. Systems

of fermentation are well recognized, much is known about their design andoperation. Another advantage is a high concentration of biomass that mayrange from 20 to 100 g d.m./L (RADMER and PARKER 1994, RUNNING et al.


Fig. 6. Fermentation tank: 1 – medium tank; 2 – nutrient dosing pomp; 3 – gas outlet; 4 – stirrer;5 – discharge of biomass

1994). The major drawbacks of this system are that it cannot be appliedto all species of algae and that the process itself poses some technologicalcomplications (BOUARAB et al. 2004, OGBONNA 1996, PEREZ-GARCIA 2011).

Conclusion

Nowadays algae are perceived as one of the types of biomass with a veryhigh energetic potential. In the technical scale, they are cultured mainly forfeedstuff or foodstuff purposes, however there are also some biorefineriesoperating in the technical scale. The exploitation of this type of substrate in thesystems of methane fermentation seems also promising, which has beenproved by results of worldwide investigations.

Translated by JOANNA MOLGA

Accepted for print 9.03.2012

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